Methods for treating cancers and restenosis with p21

ABSTRACT

The p21 gene encodes a cyclin dependent kinase inhibitor which affects cell cycle progression, but the role of this gene product in altering tumor growth has not been established. The present inventors have now discovered that the growth of malignant cells in vivo is inhibited by expression of p21. Expression of p21 resulted in an accumulation of cells in G 0 /G 1 , alteration in morphology, and cell differentiation.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention provides methods for treating or preventingrestenosis and cancer in vivo by administration of a compositioncomprising an expression vector containing a gene encoding p21 and apharmaceutical carrier.

2. Discussion of the Background

The identification of cell cycle regulatory proteins has been greatlyfacilitated by studies of mutant yeast strains with abnormalitiesrelated to cell proliferation. Among the gene products defined in yeastis Far 1 (1), whose mammalian homologue, p21, alters the activity ofcyclin-dependent kinases and is implicated in cell cycle progression andsenescence (2-13). p21, also known as WAF1, CIP1 or SDI1 (11,12,14,15),is a downstream target of the p53 tumor suppressor gene and has thusbeen implicated indirectly in malignant transformation (15-18).Induction of p53 in response to DNA damage results in G1 checkpointarrest (16-19), at which point DNA repair is accomplished prior to DNAreplication in S phase. Consistent with its presumed role as adownstream effector for p53, p21 has been shown to inhibit proliferatingcell nuclear antigen (PCNA) dependent DNA replication but not DNA repairin vitro (20).

Zhang et al, Genes & Development (1994) 8:1750) studied p21 in vitro. Asp21 functions as a kinase inhibitor, it had been predicted that normalcells should contain virtually no active cyclin kinases. Bydemonstrating that p21-containing cyclin kinases exist in both activeand inactive states; Zhang et al rationalized that p21 was involved incontrolling cell cycle progression in normal cells. Zhang et al foundthat in fibroblasts transformed with a variety of tumor viraloncoproteins, cyclin kinases exist in a binary state [cylcin/CDK];whereas in normal fibroblasts multiple cyclin kinases exist inquaternary complexes containing p21 (cyclin/CDK/proliferating cellnuclear antigen (PCNA)/p21]. Active complexes contain a single p21molecule. In contrast inactive complexes possess multiple p21 subunits.Although changes in p21 stoichiometry were sufficient to account for theconversion of active to inactive complexes in vitro, Zhang et albelieved that “association of cyclin knases with p21 must be intertwinedwith other modes of regulation in vivo,” Zhang et al noted that “it isnot known what effect association with noninhibitory levels of p21 mighthave on the function of these CDK-modifying enzymes in vivo.”

WO 94/09135 describes methods and diagnostic kits for diagnosingtransformation of a cell, involving detection of the subunit componentsof cyclin complexes. In particular, the method pertains to theinteraction of cyclins, PCNA, CDKs and low molecular weight polypeptidessuch as p21, p19 and p16.

Despite the evidence of cyclin kinase inhibitory activity in vitro, therole of p21 in tumor formation and its ability to reverse the malignantphenotype in vivo has not been defined.

SUMMARY OF THE INVENTION

Accordingly, one object of the present invention is to provide methodsfor treating and preventing cancer (tumor formation) in vivo.

A second object of the present invention is to provide methods fortreating and preventing restenosis in vivo.

A third object of the present invention is to provide methods to induceantitumor effects in cells through induction of terminaldifferentiation. This method is useful for altering expression of cellsurface proteins which might potentially facilitate immune recognitionof tumors or causing the secretion of factors which might secondarilyinhibit cell growth.

The present inventors have now determined the role of the p21cyclin-dependent kinase inhibitor on tumor cell growth and restenosis.p21 is induced by p53 (6,7,15-18) and has thus been implicated as adownstream effector of p53 tumor suppression (23). The present inventorsprovide the first direct demonstration that p21 expression is sufficientto produce these tumor and restenosis suppressor effects in vivo. p21expression was also found to facilitate transcriptional activation byNF-κB providing a mechanism whereby p21 can directly influence theexpression of genes, such as adhesion molecules, associated withdifferentiation. The suppression of tumor growth and restenosis as wellas the induction of the differentiated phenotype arises from alteredpatterns of gene expression, mediated in part by NF-κB, resulting fromp21 induced transcriptional regulation leading to terminaldifferentiation and growth arrest. Previous attempts to induce antitumoreffects through induction of terminal differentiation have involved theuse of cytotoxic drugs or hormones (25-28) which have had variablesuccess in achieving this effect.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 (A) are graphs depicting the cell cycle analysis in malignantcell lines and expression of p21 and (B) are western blots of Renca celllines transduced with adenoviral and eukaryotic expression vectors.

FIG. 2 are graphs depicting the inhibition of tumor growth followingintroduction of ADV p21 into Renca tumor cells followed by inoculation.The presence of tumor (A,C) and tumor diameter (B,D) were evaluated.

FIG. 3 are graphs depicting the effects of introduction of ADV p21 intoestablished Renca tumor cells in vivo inhibits tumor growth. Tumordiameter was measured in two perpendicular dimensions using calipers.

FIG. 4 are photographs depicting the in vitro effects of p21 onmalignant cell growth and differentiation. Phase contrast microscopy wasperformed on the indicated cells 5 days after the indicated treatments.Magnification (20×).

FIG. 5 are graphs depiciting survival of mice with established tumorstreated with ADV p21 or control vectors. BALB/c mice (a,b) or nu/nu CD-1mice (c,d) were injected with Renca cells incubated in vitro with PBS(□,▪), ADV-p21 (⋄,♦) ADV-ΔE1 (Δ,▴) at an MOI of 300.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides a method for treating cancer orrestenosis comprising administering to a patient in need thereof a tumorinhibiting amount of a composition comprising:

(i) an expression vector containing the gene which encodes p21 and

(ii) a pharmaceutically acceptable carrier.

The cDNA encoding p21 has been described by Xiong et al, Nature 366:701(1993), incorporated herein by reference.

Suitable expression vectors useful in accordance with the presentinvention include eukaryotic and viral vectors. Useful eukaryoticvectors include pRcRSV and pRcCMV or other RSV, CMV or cellularenhancers and promoters driving expression of p21 with variouspolyadenylate sequences. Preferably viral vectors are used.

Viral vector systems have been indicated as highly efficient intransferring genes to mammals containing deficient genes. See, forexample, Crystal Am. J. Med. 92(6A): 44S-52S (1992); Lemarchand et al.,Proc. Nat'l Acad. Sci. USA 89(14):6482-6486 (1992), incorporated hereinby reference. Preferably, retroviral vectors with impaired ability toreplicate and transform are used. Suitable viral vectors which expressp21 useful in accordance with the present invention include adenoviralvectors, Ad5-360 in combination with pAd-BglII as described by Davidsonet al, Nature Gen. 3:219(1993), (incorporated herein by reference).Preferably, adenoviral vectors are used.

Preferred adenoviral vectors include: ADV described by Davidson et al,Nature Gen. 3:219(1993), (incorporated herein by reference); or otheradenovirus types, including types 7001, or types 1 or 12 (as describedby Ranheim et al, J. Virol. 67:2159 (1993); Green et al, Ann. Rev.Biochem. 39:701 (1970)).

The p21 can be inserted into these expression vectors and used for celltransfection using conventional recombinant techniques such as describedby Sambrook, Fritsch, & Maniatis, in “Molecular Cloning, A LaboratoryManual” (2d ed): pp. E.5. (Cold Spring Harbor Press, Cold Spring Harbor,N.Y., 1989), the disclosure of which is hereby incorporated byreference. Alternatively, the expression vectors can be prepared usinghomologous recombination techniques as described by Davidson et al,1993, Nature Gen. 3:219-223 or Lemarchand et al. Proc. Nat'l Acad. Sci.USA 89(14):6482-6486 (1992).

The expression vectors of the present invention can additionally containregulatory elements such as promoters and selection markers such asantibiotic resistance genes.

It is well established that viral vectors will be taken up in andintegrated into cells in vivo and express the viral DNA, includinginserted constructs. See, e.g., Yoshimura et al. J. Biol. Chem.268(4):2300-2303 (1993); Crystal Am. J. Med. 92(6A):445-525 (1992);Lemarchand et al. Proc. Nat'l Acad. Sci. USA 89(14):6482-6486 (1992) thedisclosures of which are hereby incorporated by reference.

In an alternate embodiment, it is also understood that other deliverysystems besides expression vectors can be used to deliver p21 protein.Principally, these techniques, including the use of liposomes and DNAconjugates, are expected to provide similar delivery yields as thoseprovided by the expression vectors discussed above. That is, rather thanexpressing the p21 gene via an expression vector, it is also possible toincorporate a therapeutic amount of p21 in a vehicle.

In a second alternate embodiment, p21 can be expressed as a fusionprotein. In this embodiment, the gene encoding p21 is fused to a geneencoding an immunotherapeutic agent, genetic therapeutic (such asHLA-B7), protein (such as cytokines, preferably, GM-CSF, IL-2 and/orIL-12), prodrug converting enzymes (such as thymidine kinase, cytosinedeaminase and β-glucurodinase) or anticancer drug such as cis-platinum.

Fusion genes are proteins produced therefrom are described in MolecularCloning: A Laboratory Manual, Sambrook et al, 2nd edition, Cold SpringHarbor Laboratory Press, 1989 (in particular, chapter 17) incorporatedherein by reference.

Thymidine kinase can be obtained as described in AU8776075, incorporatedherein by reference. β-glucuronidase and fusion proteins thereof aredescribed in U.S. Pat. No. 5,268,463 and U.S. Pat. No. 4,888,280,incorporated herein by reference. Cytosine deaminase and fusion proteinsthereof are described in WO 9428143, incorporated herein by reference.

In addition combination therapies of viral vectors and liposomes havealso shown tremendous promise and are also contemplated for use in theinvention. Yoshimura et al, J. Biol. Chem., 268(4):2300-2303 (1993),incorporated herein by reference.

Liposomes are known to provide highly effective delivery of activeagents to diseased tissues. For example, pharmacological or otherbiologically active agents have been effectively incorporated intoliposomes and delivered to cells. Thus, constructs in accordance withthe present invention can also be suitably formed in liposomes anddelivered to selected tissues. Liposomes prepared from cationic lipids,such as those available under the trademark LIPOFECTIN (LifeTechnologies, Inc., Bethesda, Md.) are preferred. Particularly appealingto liposome based treatments is the fact that liposomes are relativelystable and possess relatively long lives, prior to their passage fromthe system or their metabolism. Moreover, liposomes do not raise majorimmune responses.

Thus, in one aspect of the present invention a vector containing a geneencoding p21 is incorporated into a liposome and used for the deliveryof the construct to a specific tissue. The liposome will aid theconstruct in transfecting a cell and becoming expressed by the cell,ultimately generating p21 protein.

The composition of the present invention is a therapeutically effectiveamount of a vector which expresses p21 and a pharmacuetically acceptablecarrier. In order to administer the viral vectors, suitable carriers,excipients, and other agents may be incorporated into the formulationsto provide improved expression of p21.

A multitude of appropriate formulations can be found in the formularyknown to all pharmaceutical chemists: Remington's PharmaceuticalSciences, 15th Edition (1975), Mack Publishing Company, Easton, Pa.18042. (Chapter 87: Blaug, Seymour). These formulations include forexample, powders, pastes, ointments, jelly, waxes, oils, lipids,anhydrous absorption bases, oil-in-water or water-in-oil emulsions,emulsions carbowax (polyethylene glycols of a variety of molecularweights), semi-solid gels, and semi-solid mixtures containing carbowax.

Any of the foregoing formulations may be appropriate in the treatmentwith the viral vectors, provided that the viral particles areinactivated in the formulation and the formulation is physiologicallycompatible.

The amount of p21 to be administered will depend on the size of thepatient and the state to which the cancer has progessed. By modifyingthe regulatory elements of the vector using conventional techniques orby varying the amount of viral vector titre administered, the amount ofp21 expression can be adjusted to the patients needs. Typically, it isdesirable to deliver approximately 50 viral vectors per cell to betreated. With the adenovirus, formulations should generally contain onthe order of 10¹⁰ viral infectious units per ml. With retrovirus,slightly different titers may be applicable. See Woo et al, Enzyme38:207-213 (1987), incorporated herein by reference. Additionalassistance in determining appropriate dosage levels can be found in Kayet al, Hum. Gene Ther. 3:641-647 (1992); Liu et al, Somat. Cell Molec.Genet. 18:89-96 (1992); and Ledley et al, Hum. Gene Ther. 2:331-358(1991), incorporated herein by reference.

Depending upon the particular formulation that is prepared for theadministration of the expression vectors, administration of thecompositions of the present invention can be accomplished through avariety of methods. The composition of the present invention arepreferably administered by direct injection of the expression vector (orliposome containing the same) into the tumor such as described in U.S.Pat. No. 5,328,470, incorporated herein by reference.

Breast, renal, melanoma, prostate, glioblastoma, heptocarcinoma, colonand sarcoma cancer types can be treated in accordance with the presentinvention. Methods of diagnosis and monitoring these cancer types arewell known in the art.

Arterial injury from angioplasty induces a series of proliferative,vasoactive, and inflammatory responses which can lead to restenosis.Although several factors have been defined which stimulate this processin vivo, the role of specific cellular gene products in limiting theresponse is not well understood. The present inventors have now foundthat p21 acts to limit the proliferative response to balloon catheterinjury. Vascular endothelial and smooth muscle cell growth was arrestedthrough the ability of p21 CKI to inhibit cyclin-dependent kinases andprogression through the G₁ phase of the cell cycle. Restenosis is aclinical condition which can be diagnosised and monitored as describedin Epstein et al, JACC 23(6):1278 (1994) and Landau et al, MedicalProgress 330(14):981 (1994), incorporated herein by reference.

The compositions of the present invention can be used to treat allmammals, in particular humans.

The compositions of the present invention can be administered incombination with immunotherapeutic agents, genetic therapeutics (such asHLA-B7), proteins (such as cytokines, preferably, GM-CSF, IL-2 and/orIL-12), prodrug converting enzymes (such as thymidine kinase, cytosinedeaminase and β-glucurodinase) and anticancer drugs such ascis-platinum. Alternatively, the compositions of the present inventioncan be administered in combination with expression vectors comprisinggenes encoding the above immuno-therapeutics, genetic therapeutics,proteins, prodrug converting enzymes and anticancer drugs.

Alternatively, the compositions can be administered during adoptive celltransfer therapy.

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples which are providedherein for purposes of illustration only and are not intended to belimiting unless otherwise specified.

EXAMPLES Example 1 Use of P21 Cycin-Dependent Kinase Inhibitor to toTreat Restenosis In Vivo

In this study, the effect of p21 expression on endothelial and smoothmuscle cells in vitro and in a porcine model of arterial balloon injuryin vivo was analyzed.

Cell Culture and Transfection

Primary porcine vascular endothelial and smooth muscle cells werederived from the aorta of 6-month-old domestic Yorkshire pigs and wereused between the second and fifth passage. Endothelial and smooth musclecells were grown to 70% confluence in medium 199 with 10% FBS. Cellswere infected with ADV-p21 or ADV-ΔE1 (MOI 300/cell) for 1 hour in DMEMand 2% FCS, and normal media was added after 1 hour. Control cells wereuninfected and carried in M199 with 10% FBS. Twenty-four hours later,the cells were split into 6 well dishes at 6×10⁴ cells per well. Cellswere harvested at 0, 2, 5, 7, and 10 days, and cell numbers weredetermined by a hemocytometer. Cell viability was assessed by trypanblue exclusion.

Cell Cycle Analysis

Cells were infected at an MOI of 300/cell with the ADV-ΔE1 or ADV-p21vectors as described above, harvested, washed with PBS twice, and thenfixed in 70% ethanol (EtOH) (King et al, Cell 79, 563-571 (1994)) for 30minutes at 4° C. The cells were treated with 1U DNase-free RNase in 1 mlof PBS for 30 minutes at 37° C., and resuspended in 0.05 mg/ml propidiumiodide (made as a 10× stock in PBS). Cells were analyzed by flowcytometry using a FACScan model (Becton Dickinson). Fluorescencemeasurements were accumulated to form a distribution curve of DNAcontent. Fluorescence events due to debris were substracted beforeanalysis.

Adenoviral Vectors

The recombinant adenoviral vector, ADV-p21, was constructed byhomologous recombination between sub360 genomic DNA, an Ad5 derivativewith a deletion in the E3 region, and a p21 expression plasmid, pAd-p21.Briefly, the pAd-p21 plasmid was prepared by introducing the HindIII-XbaI fragment of a p21 expression vector utilizing the Rous sarcomavirus promoter (RSV) to regulate expression of p21 into the Bgl II siteof pAd-Bgl II (Heichman & Roberts, Cell 79, 557-562 (1994)). Thestructure of these replication defective E1A, E1B deleted viruses wasconfirmed by Southern blotting. All recombinant viruses were propagatedin 293 cells and purified as described (Davidson et al, 1993, NatureGen. 3:219-223). Cesium chloride purified virus was dialysed againstPBS, and diluted for storage in 13% glycerol-PBS solution to yield afinal concentration of 1-3×10¹² viral particles/ml (0.8-5×10¹⁰ pfu/ml).All stocks were sterilized with a 0.45 μm filter and evaluated for thepresence of replication competent adenovirus by infection at, a MOI of10 onto 3T3 cells. None of the stocks used in these experiments yieldedreplication-competent virus.

Porcine Arterial Injury

After anesthesia and intubation, domestic Yorkshire pigs (12-15 kg)underwent sterile surgical exposure of the iliofemoral arteries, and adouble-balloon catheter (C. R. Bard, Inc.) was inserted into theiliofemoral artery. The proximal balloon was inflated to a pressure of500 mmHg, measured by an on-line pressure transducer, for 5 minutes.Animals were sacrificed 1, 7, and 21 days after injury.

In Vivo Gene Transfer

Direct gene transfer was performed in the iliofemoral arteries ofYorkshire pigs using a double balloon catheter as described (Nabel etal, 1990, Science 249:1285-1288). In each animal, both iliofemoralarteries were infected with the same vector at a titer of 1×10¹⁰ pfu/ml,and 0.7 ml was used in each animal (final dose of 7×10⁹ pfu) (Ohno etal, 1994, Science 265:781-784; Chang et al, 1995, Science 267:518-522).

The vessel segments infected with ADV-p21 (n=28 arteries) or ADV-ΔE1(n=28 arteries) vectors were excised 7 or 21 days later. To evaluateintimal cell proliferation, animals sacrificed at 7 days received anintravenous infusion of 5-bromo-2′-deoxycytosine (BrdC) (Sigma, St.Louis, Mo.) 25 mg/kg total dose, 1 hour prior to death. Each artery wasprocessed in an identical manner as described (Ohno et al, 1994, Science265:781-784). All animal experiments were performed in accordance withNIH guidelines and with approval of the University of Michigan Committeein the Use and Care of Animals.

RT-PCR Analysis

Total RNA was prepared using Trizol reagents (GIBCO/BRL) according tothe manufacturer's protocol. Briefly, artery samples were homogenized inTrizol reagent. RNA was precipitated with ethanol (EtOH), washed in cold75% EtOH three times, dried and resuspended in RNAse-free TE buffer. PCRfor the p21 gene was performed (Muller et al, 1994, Circ. Res.75:1039-1049) in the presence or absence of reverse transcriptase (RT)with the primers: 5′-GAG ACA CCA CTG GAG GGT GAC TTC G-3′ (sense); and5′-GGG CAA ACA ACA GAT GGC TGG CAA C-3′ (antisense). The antisenseprimer was specific for recombinant p21 RNA and not endogenous porcinep21 RNA.

Measurement of Cell Proliferation and Morphometry

Measurements of cell proliferation were made 7 days after balloon injuryand adenoviral infection using a monoclonal antibody to BrdC. Arterialsections were fixed, embedded, and sectioned, and immunohistochemistryusing a monoclonal anti-5-bromo-2′-deoxycytidine antibody was performed(Ohno et al, 1994, Science 265:781-784) to label nuclei in proliferatingcells. For each artery, the number of labeled and Unlabeled nuclei inthe intima were quantitated using a microscope based video imageanalysis system (Image One Systems, Universal Imaging Corporation,Westchester, Pa.). A proliferation index was calculated as the ratio oflabeled cells to total number of cells.

Intimal and medial cross sectional areas were measured in 4 sectionsfrom each artery spanning the 2 cm region of arterial injury andadenoviral infection with the image analysis system (Ohno et al, 1994,Science 265:781-784). An intima to media (I/M) area ratio for eachartery was determined as the average I/M area ratio of the 4 sections.

Immunohistochemistry

Immunohistochemical studies were performed with antibodies to BrdC,smooth muscle α-actin, and p21, using methods as described (Ohno et al,1994, Science 265:781-784; Muller et al, 1994, Circ. Res. 75:1039-1049).The following primary antibodies were used: a monoclonal mouse anti-BrdCantibody, 1:1000 dilution (Amersham Life Sciences); a monoclonal mouseanti-smooth muscle a actin antibody, 1:500 dilution (Boehringer MannheimBiochemical); and a polyclonal mouse anti-human p21 antibody, 1:1500dilution (Santa Cruz). Control experiments were performed using apurified mouse IgG_(2b) antibody, 1:100 dilution (Promega), which didnot stain the arterial specimens. Slides were developed with either astreptavidin-horseradish peroxidase complex (Vector Laboratories) or aVectastain ABC-alkaline phosphatase reagent (Vector Laboratories), andcounterstained in methyl green.

Statistical Analysis

Comparisons of intimal BrdC labeling index and I/M area ratios betweenADV-p2l and ADV-ΔE1 arteries were made by two-tailed, unpaired t-test.Statistical significance was assumed if a null hypothesis could berejected at the 0.05 level.

Results

Expression of p21 Inhibits Vascular Cell Proliferation and Induces CellCycle Arrest In Vitro.

To study the effects of p21 on vascular cell growth and cell cycledistribution, quiescent porcine vascular endothelial and smooth musclecells were infected in vitro with an adenoviral vector, ADV-p21 or acontrol vector containing an E1 deletion, ADV-ΔE1 and then stimulated toproliferate by incubation in 10% FBS. Exposure of uninfected or ADV-ΔE1infected cells to serum resulted in rapid proliferation of endothelialand smooth muscle cells. In contrast, expression of p21 in vascularendothelial and smooth muscle cells resulted in inhibition of cellproliferation by >90%; these cells were still-viable (>95%) as assessedby trypan blue exclusion. Expression of p21 in vascular endothelial andsmooth muscle cells also resulted in accumulation of cells in G₀/G₁, asassessed by propidium iodine staining. These data suggest that cellswere arrested in cell cycle by p21 expression rather than p21 causingcell death.

p21 is Induced in Balloon Injured Arteries In Vivo.

To investigate the potential of p21 to regulate vascular cell growth invivo, we first determined whether p21 expression is induced in injuredarteries. Porcine iliofemoral arteries were either uninjured or injuredby balloon angioplasty, and injured segments were analyzed 1, 7, and 21days later for p21 expression, assessed by immunohistochemistry with ap21 antibody. This porcine model of arterial injury results in intimalthickening by 3 weeks (Ohno et al, 1994, Science 265:781-784). Thelesion is characterized by rapid smooth muscle cell proliferation duringthe first 7 days after arterial injury, followed by expansion of theintima due to elaboration of extracellular matrix during the subsequent2 weeks. Normal, uninjured porcine arteries expressed no p21. One dayfollowing arterial injury, p²1 protein was not present in the intima;however, at 7 days, there was p21 protein in approximately 50% ofintimal smooth muscle cells. At 21 days, p21 expression was present inlower regions of the intima, next to the internal elastic lamina, inregions where cell proliferation was not present (Ohno et al, 1994,Science 265:781-784). Indeed, p21 expression in general was inverselycorrelated with smooth muscle cell proliferation. These findings suggestthat p21 expression is associated with arrest of vascular cellproliferation in injured arteries.

Expression of p21 in Injured Arteries Limits the Development of IntimalHyperplasia.

To assess the direct effect of p21 on vascular cell growth in vivo, p21vectors were introduced into porcine arteries immediately followinginjury. The right and left iliofemoral arteries of domestic pigs wereballoon injured and infected with ADV-p21 or ADV-ΔE1 using adouble-balloon catheter (1×10¹⁰ pfu/ml, 0.7×10¹⁰ pfu total dose). Invivo gene transfer of ADV-p21 was demonstrated in injured porcinearteries 7 days after infection by RT-PCR analysis. p21 RNA was detectedby RT PCR in infected left and right iliofemoral arteries but not in anoninfected carotid artery from the same animal or in ADV-ΔE1noninfected and infected arteries.

The effect of p21 expression on intimal cell growth in vivo was nextassessed by two methods, quantitating incorporation of BrdC into intimalcells 7 days after gene transfer and measuring I/M area ratios at 3weeks. A 35% reduction in intimal BrdC incorporation was observed inADV-p21 infected arteries, compared with ADV-ΔE1 arteries, 7 days aftergene transfer (5.3±0.9% vs. 8.1±0.4%, p=0.035). These BrdC labeledintimal cells costained with a monoclonal antibody to smooth muscleα-actin, suggesting that inhibition of intimal smooth muscle cellproliferation was present in ADV-p21 animals. A significant reduction inI/M area ratio of 37% was observed in ADV-p21 infected arteries,compared with ADV-ΔE1 infected arteries (0.37±0.06 vs. 0.59±0.06,p=0.015). These results suggest that infection of arteries with ADV-p21at the time of balloon injury inhibits the proliferation of intimalsmooth muscle cells and significantly limits the development of aneointima.

Example 2 Use of P21 Cycin-Dependent Kinase Inhibitor to SuppressTumorigenicity In Vivo

In this study, the effect of p21 expression on tumor growth in vitro andin a murine model in vivo was analyzed.

Cell Cycle Analysis

Cells were infected at an MOI of 200-300 with the ADV-ΔE1 or ADV-p21vectors or transfected with the p21 expression vector by DNA/liposomecomplexes. The cells were infected as above and harvested, washed withPBS twice, then fixed in 70% EtoH for 30 minutes of 4° C. The cells weretreated with 1U Dnase-free RNase in 1 ml of PBS for 30 minutes at 37°C., and finally, resuspended in 0.05 mg/ml propidium iodide (made as a10× stock in PBS, and cells were analyzed by flow cytometry using aFACScan model (Becton Dickinson). Fluorescence measurements wereaccumulated to form a distribution curve of DNA content. Fluorescenceevents due to debris were substracted before analysis.

Western Blot Detection of p21

3-5×10⁶ cells were harvested at the time points indicated, lysed with 1ml of 50 mM Tris-Hcl (pH 6.8), 100 mM DTT, 2% SDS, 0.1% bromophenolblue, 10% glycerol, and boiled for 5 minutes. The samples were finallyspun at 10,000 rpm for 5 minutes, and supernatants were collected. 20 μlwere loaded into 15% SDS-PAGE and blotted into nitrocellulose membrane.p21 protein was visualized using an antipeptide rabbit polyclonalantibody (Santa Cruz) together with an antirabbit horseradish peroxidasesecondary antibody and subsequent ECL chemiluminescent detection(Amersham).

Gene Transfer of p21

Cells were maintained in Dulbecco's modified eagle medium (DMEM)containing 10% fetal calf serum. The recombinant adenoviral vector,ADV-p21, was constructed by homologous recombination between sub360genomic DNA, an Ad5 derivative with a deletion in the E3 region, and ap21 expression plasmid, pAd-p21. These recombinant adenoviral vectorshave sequences in the E1A and E1B region deleted, impairing the abilityof this virus to replicate and transform nonpermissive cells. Briefly,the pAd-p21 plasmid was prepared by introducing the Nru I and Dra IIIfragment from pRc/CMV-p21, kindly provided by Drs. D. Beach and G.Hannon (Xiong et al, Nature 366, 701 (1993); Serano et al, Nature 366,704 (1993)) into the Bgl II site of pAd-Bgl II (Davidson et al, NatureGenet. 3, 219 (1994)) which had the left hand sequence of Ad5 genome,but not E1A and E1B. Virus was prepared as described previously (Ohno etal, Science 265, 781 (1994). The structure of these viruses wasconfirmed by Southern blotting. All recombinant viruses were propagatedin 293 cells and purified as described (Davidson et al, Nature Genet. 3,219 (1994)). Cesium chloride purified virus was dialysed against PBS,and diluted for storage in 13% glycerol-PBS solution to yield a finalconcentration of 1-3×10¹² viral particles/ml (0.8-5×10¹⁰ pfu/ml). Allstocks were sterilized with a 0.45 μm filter and evaluated for thepresence of replication competent adenovirus by infection at a MOI of 10onto 3T3 cells. None of the stocks used in these experiments yieldedreplication-competent virus.

The eukaryotic expression plasmid, pRc/RSV p21, was prepared byintroduction of the p21 cDNA from pRc/CMV-p21 into pRc/RSV (Invitrogen),and transfection of 293 cells performed by using calcium phosphatetransfection (Perkins et al., manuscript submitted).

Bystander Assay

U373 human glioblastoma cells, kindly provided by Dr. K. Murazko, wereinfected with ADV-p21 (MOI 200). One day later, cells were trypsinized,counted, and mixed with the indicated number of uninfected U373 cells.10,000 cells for each mixed population were plated into a 96 well disk.Five days later, the MTT assay (Mosman, J. Immunol. Methods 65, 55(1983)) was performed to determine the proliferation rate of these cellpopulations.

Gene Transfer of p21 and Effect on Cell Cycle Progression in MalignantCells.

The effect of p21 on cell cycle distribution was determined in tumorcell lines by infection with an adenoviral vector, ADV-p21, or a similarEl deletion virus with no recombinant p21, ADV-ΔE1. Expression of p21 inthe adenoviral vector was regulated by the CMV enhancer/promoter andbovine growth hormone polyadenylation sequence. Expression of p21 withina representative malignant cell line, the B16BL6 melanoma, resulted inan accumulation of cells in the G₀/G₁ phase of the cell cycle,suggesting arrest predominantly at the G1/S boundary (FIG. 1 a).Recombinant p21 expression was confirmed in murine (Renca) or human(293) renal cell-carcinoma lines, and the murine (B16BL6) melanoma cellline by using Western blot analysis. Readily detectable proteinexpression from the adenoviral vector was achieved −1 day afterintroduction of the gene (FIG. 1 b, lanes 4,5,13,14 vs. 1-3,10-12). Inaddition, a eukaryotic expression plasmid regulated by the Rous sarcomavirus (RSV) enhancer/promoter and bovine growth hormone polyadenylationsite showed comparable expression in 293 cells (FIG. 1 b, lanes 7,9 vs.6,8). In both cases, expression of the recombinant protein correlatedwith inhibition of cell division and other vectors with the sameregulatory elements did not show the effects of p21 described here.

Differentiation and Morphologic Effects of p21.

When the effect of p21 on cell growth was examined in vitro, tumor cellsinfected with ADV-p21 showed morphological changes, such as an increasednuclear to cytoplasmic ratio, an increase in adherence and growtharrest, consistent with a differentiated phenotype (FIGS. 2,3). Humanmelanoma cells, UM-316, showed nuclear condensation and a >4-foldincrease in melanosome formation by electron microscopy after infectionwith ADV-p21 (FIG. 2; p<0.005 by the Wilcoxon rank sum test). In thesecells, an ˜5-fold increase in melanin production was observed within 2days after gene transfer in cells and supernatant fractions in vitro(FIG. 3).

In some lines, cell death was observed to follow terminaldifferentiation after extended cell culture, but there was no evidenceof apoptosis, as determined by the pattern of DNA fragmentation (FIG. 4a), propidium iodine staining or TdT immunostaining. In addition,mixtures of uninfected and infected cells showed a lack of bystandereffect (FIG. 4 b), suggesting that gene transfer and expression inrecipient cells was required and that efficient infection of p21 isrequired to eradicate growth of established tumors.

Inhibition of Tumor Cell Growth In Vivo.

To assess the effect of p21 on the growth of malignant cells in vivo,Renca cells were infected with ADV-p21, an ADV-ΔE1 control, or incubatedwith phosphate buffered saline (PBS), and inoculated into recipientmice. p21 expression completely suppressed the growth of tumors in allanimals inoculated with 2×10⁵ cells (FIGS. 5 a,b). Because it remainedpossible that expression of p21 could alter the immunogenicity ofinfected cells and thus work through an immune mechanism, similarstudies were undertaken in CD-1 nu/nu immunodeficient mice. Similarinhibition of tumor growth was observed in these animals (FIGS. 5 c,d),consistent with a direct effect on cell proliferation.

To determine whether ADV-p2.1 could alter the growth of establishedtumors, Renca tumor nodules (˜0.5 cm) were injected with either PBS,ADV-ΔE1, or ADV-p21. Direct transfer of adenoviral vectors encoding ahuman placental alkaline phosphatase reporter into established tumorscaused infection of up to 95% of cells estimated by quantitativemorphometry after 5 repeated daily injections of 10⁹ PFU. This treatmentalso inhibited tumor growth, and when injections were performedrepetitively (5 daily injections, repeated after one week), could leadto long-term cure as determined by survival (>40 days) and the inabilityto detect macroscopic tumor in mice with previously detectable nodules.In both cases these results were statistically significant.

REFERENCES

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Having now fully described the invention, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the inventionas set forth herein.

1-16. (canceled)
 17. A kit for treating a disease in a patient, the kitcomprising a syringe and a nucleic acid comprising a gene encoding p21.18. The kit of claim 17, further comprising a pharmaceutical carrier.19. The kit of claim 18, wherein the pharmaceutical carrier comprisesthe nucleic acid.
 20. The kit of claim 17, wherein the nucleic acid isan expression vector.
 21. The kit of claim 20, wherein the expressionvector comprises a viral promoter.
 22. The kit of claim 21, wherein theviral promoter is a CMV promoter.
 23. The kit of claim 21, wherein theviral promoter is a RSV promoter.
 24. The kit of claim 17, wherein aviral particle comprises the nucleic acid.
 25. The kit of claim 24,wherein the viral particle is an adenovirus particle.
 26. The kit ofclaim 24, wherein the viral particle is a retrovirus particle.
 27. Thekit of claim 17, further comprising a liposome.
 28. The kit of claim 27,wherein the liposome comprises the nucleic acid.
 29. The kit of claim17, wherein the nucleic acid comprises a second gene.
 30. The kit ofclaim 29, wherein the second gene encodes HLA-B7, an immunotherapeuticagent, cytokine, or prodrug converting enzyme.
 31. The kit of claim 30,wherein the prodrug converting enzyme is thymidine kinase.
 32. The kitof claim 29, wherein the gene encoding p21 and the second gene areoperatively linked.
 33. The kit of claim 32, wherein the gene encodingp21 and the second gene are operatively linked such that they encode afusion protein.
 34. The kit of claim 33, wherein the fusion protein is ap21-thymidine kinase fusion protein.